1. Field of the Invention
The present invention relates generally to the field of molecular biology and muscle physiology. More specifically, this invention relates to the MyoD1 gene, the methylation pattern within the 5' upstream region of this gene, and diagnostic and therapeutic applications thereof.
2. Description of the Related Art
Rhabdomyosarcoma is the most common soft-tissue malignancy in childhood, accounting for 4 to 8% of all pediatric cancers (Enzinger and Weiss, 1995). Rhabdomyosarcomas are a heterogeneous group of malignancies characterized by varying degrees of differentiation, ranging from uncommitted primitive mesenchymal cells to fetal myotubes (Parham, 1995). Because of the clinical heterogeneity and the primitive histologies, it is often a challenging task to diagnose poorly differentiated rhabdomyosarcomas from other small round cell tumors.
Within the class of pediatric rhabdomyosarcomas, there are two major subtypes, embryonal and alveolar, which present with biologically and clinically distinct behaviors. The botryoid and the spindle cell variants of embryonal rhabdomyosarcoma are associated with favorable prognosis (Leuschner et al, 1993), whereas alveolar rhabdomyosarcomas usually have a more aggressive clinical behavior and are associated with a worse outcome than all embryonal variants (Tsokos et al, 1985). The molecular events that are involved in the development of a rhabdomyosarcoma are still poorly understood. The discovery of the MyoD gene family, however, has provided insight into the regulation of myogenesis and has prompted the search for applicable markers that can assist in the clinical diagnosis of rhabdomyosarcomas (Weintraub et al, 1991).
The genes of the MyoD family create the nodal point of early myogenesis by upregulating the expression of myogenic genes such as desmin, creatine kinase, and myosin (Weintraub et al, 1991). Members of the MyoD family, including MyoD1, myogenin, myf-5, and myf-6, are characterized by a helix-loop-helix motif with an adjacent basic domain (bHLH), and are part of a large class of transcription activators (Braun et al, 1989; Davis et al, 1987; Rhodes, 1989; Wright et al, 1989). Arbitrary expression of these genes causes initiation of myogenesis not only in primitive mesenchymal cells but also in differentiated non-muscle cells such as fibroblasts (Weintraub et al, 1991).
Among these genes, the MyoD1 has been the most studied in rhabdomyosarcoma. The MyoD1 gene product functions as a homodimer or heterodimer by binding to the protein product of the E2A gene, causing transcription activation (Davis et al, 1990), or to the Id protein, causing transcription inhibition (Li and Olsen, 1992). In normal myogenesis, as occurs in growth and repair, the expression of MyoD1 initiates a cascade of events leading to the formation of a myotube, and is subsequently suppressed (Montarras et al, 1991; Ott et al, 1991). In rhabdomyosarcoma, the molecular events that cause the inhibition of MyoD1 expression are distorted or lost, so that the expression of MyoD1 persists at high levels in tumor cells (Tapscott et al, 1993). As a result, the MyoD1 protein has been recognized as a sensitive and specific marker for both childhood and adult rhabdomyosarcomas.
Abnormal patterns of DNA methylation are thought to play an important role in the development of cancer by altering gene expression and causing genomic instability (Bird, 1996). De novo methylation of the human MyoD1 gene has been observed in a number of neoplasms, including colorectal cancer (Iacopetta et al, 1997), breast carcinomas (Hahnel et al, 1996), and ovarian carcinomas (Cheng et al, 1997). In those studies, however, the tumors were of non-muscle origins where the MyoD1 gene is not expressed due to unknown silencing mechanisms. Therefore, the relationship between hypermethylation of certain MyoD1 regions and tumor development was unclear. In addition, a mouse or a human MyoD1 cDNA probe was used that would only allow detection of methylation alterations in the coding region but not the 5' upstream region where the transcriptional promoter is usually located.
MyoD1 has been implicated in the control of proliferation in both normal cells and tumors, independent of its role in the activation of the myogenic differentiation program (Crescenzi et al, 1990, Sorrentino et al, 1990). De novo methylation of the murine MyoD1 upstream CpG island occurs during the establishment of immortal cell lines (Jones et al, 1990), and progressive increases in the methylation status and heterochromatization of the CpG island was found during oncogenic transformation (Rideout et al, 1994). However, these two findings are thought to be in vitro phenomena related to cell culturing situations, because the mouse MyoD1 gene was found unmethylated in skeletal muscle and non-muscle cells which do not express the gene. Of interest has been the observation that patchy, heterogeneous expression of MyoD1 is frequently observed in embryonal rhabdomyosarcoma, whereas strong diffuse positivity is usually observed in alveolar rhabdomyosarcomas. This phenomenon was also reflected by Northern blot analysis in a study by Scrable et al. which indicated that higher levels of MyoD1 mRNA transcripts are produced in alveolar rhabdomyosarcomas as compared to tumors of the embryonal subtype (Scrable et al., 1989).
The human MyoD1 gene is mapped to chromosome 11p15.4 (Scrable et al., 1990), adjacent to a number of imprinted genes including IGF2, H19, and p.sub.57.sup.KIP, all of which have shown imprinting disturbances leading to aberrant gene expression associated with abnormal development and cancer (Ogawa et al., 1993; Taniguchi et al., 1997; Taniguchi et al., 1995). Loss of heterozygosity for chromosome 11p15, together with loss of imprinting for genes in this region, is found frequently in embryonal rhabdomyosarcomas (Ohlsson et al., 1993; Scrable et al., 1989). Overexpression of the MyoD1 and IGF2 are both found in the embryonal subtype of rhabdomyosarcoma, which occurs with a n increased incidence in Beckwith-Wiedemann syndrome (Dias et al, 1990; Ohlsson et al, 1993; Sotelo-Avila and Gooch, 1976). On the other hand, hypermethylation at CpG islands of tumor suppressor genes is known to silence their expression in tumorigenesis, as demonstrated for the VHL gene in renal tumors (Herman et al., 1994) and the p16.sup.INK4A in a variety of malignancies (Ng et al., 1997).
Alveolar rhabdomyosarcomas, on the other hand, are characterized by chromosomal translocations t(2;13) or t(1;13), which respectively generate the Pax3-FKHR or the Pax7-FKHR fusion genes (Barr et al., 1993; Davis et al., 1994). Two recent studies on mouse skeletal myogenesis suggested that Pax3, an evolutionarily conserved transcription factor expressed in the lateral dermomyotome, may control myogenesis either by directly activating the transcription of the MyoD1 gene (Tajbakhsh et al, 1997), or by mediating the transcriptional activation of MyoD1 and Myf-5 in response to muscle-inducing signals (Maroto et al, 1997). In humans, a chromosomal translocation t(2;13) juxtaposes the amino terminal DNA binding domains of PAX3 with the transcriptional activation domain of FKHR (a Forkhead family member) in alveolar rhabdomyosarcoma (Galili et al. 1993). The tumor-specific Pax3-FKHR fusion protein is a more potent transcription activator than the wild-type Pax3 protein (Sublett et al, 1995), suggesting that a gain-of-function of Pax3 may therefore be involved in the etiology of alveolar rhabdomyosarcoma. This argument is further strengthened by the identification of a less frequent translocation t(1;13) in alveolar rhabdomyosarcoma which rearranges PAX7, another member of the Pax family, to generate a Pax7-FKHR chimera, analogous to the Pax3-FKHR fusion (Davis et al, 1994). Therefore, it appears that there is a relationship between the high level of MyoD1 expression and the characteristic Pax3-FKHR or Pax7-FKHR fusion products in alveolar rhabdomyosarcoma.
The identification of a potential Pax3 consensus binding site in the promoter region of the MyoD1 gene (-578 through -569) suggests that Pax3 may activate the MyoD1 transcription by directly binding to the MyoD1 promoter. A similar Pax3 binding site has been identified in the promoter region of the c-Met proto-oncogene, which is expressed in limb muscle progenitors and is required in the mouse for the limb muscle development (Epstein et al, 1996). Pax3 binding site in the c-Met promoter may contribute to direct transcription regulation by Pax3 (Epstein et al, 1996). However, unlike MyoD1 expression, which is found in most rhabdomyosarcomas and establishes the diagnosis of this tumor type, c-Met expression is less consistently found in rhabdomyosarcoma cell lines, and is unlikely to account for the Pax3-FKHR or Pax7-FKHR tumorigenicity. In addition, the Pax3-FKHR or Pax7-FKHR fusion proteins in alveolar rhabdomyosarcoma have been shown to bind similar nuclear DNA targets but are more potent transcription activators than the wild-type Pax3 or Pax7 (Sublett et al, 1995). Therefore, the Pax3-FKHR or Pax7-FKHR fusion proteins might be responsible in part for the enhanced transcription activation of the MyoD1 gene in alveolar rhabdomyosarcoma, presumably by binding to the Pax3 consensus site in the promoter region of the MyoD1 gene.
The MyoD1 gene consists of three exons, and is highly conserved in mammalian species. The mouse MyoD1 gene has a relatively large CpG island that spans the first exon and extends a short distance both 5' and 3' from the first exon (Rideout et al, 1994). In mouse, methylation has been shown to play a role in preventing MyoD1 expression in cultured non-muscle cells. Demethylation of the MyoD1 gene initiates the myogenic program in murine fibroblasts (Konieczny and Emerson, 1984), whereas methylation of the MyoD1 promoter region may turn off MyoD1 expression both in myoblasts and in fibroblasts (Zingg et al, 1991). However, a correlation between the MyoD1 gene methylation status and its level of expression has not been demonstrated. The human MyoD1 mRNA was cloned in 1991 (Pearson-White, 1991).
Thus, the prior art is deficient in the understanding of how the MyoD1 gene is regulated during normal myogenesis and rhabdomyosarcoma and the effect methylation of the MyoD1 gene has on gene expression. The prior art is also deficient in the ability to accurately diagnose rhabdomyosarcoma from other small round cell pediatric tumors and to differentiate the alveolar subtype of rhabdomyosarcoma from the embryonal subtype. The present invention fulfills these long-standing needs and desires in the art.